Polymerase Chain Reaction (PCR) is a powerful technique commonly used in today's laboratories for specific amplification and detection of as little as a single copy of a target nucleic acid sequence. PCR also is used for the quantification of nucleic acid sequences because of the quantitative relationship between the amount of starting target sequence and the amount of PCR product at any given cycle.
End-point PCR is widely used in applications for amplifying nucleic acid templates. In end-point PCR, a template is added at the beginning of a PCR reaction and the reaction is carried out in multiple cycles, usually 20 to 50 cycles. It is the end product of the amplification reaction which is detected and/or quantified. In contrast, real time quantitative PCR (QPCR) monitors the progress of a PCR amplification as it is occurring. In real-time QPCR techniques, signals (generally fluorescent) are monitored as they are generated. The number of cycles required to achieve a chosen level of fluorescence varies directly with the concentration of amplifiable targets at the beginning of the PCR process, enabling a measure of fluorescent intensity to provide a measure of the amount of target DNA in a sample. Fluorescence intensities are detected during the annealing/extension period of each PCR cycle and the output of this detection is fed to a processor for storage and data manipulation. End-point PCR is less accurate than QPCR because the measurement is made later in the PCR, which means more variables have had the opportunity to affect the results. For example, as reaction components are depleted, amplification is reduced. Different signal levels at endpoint might be caused by slight differences in limiting reagents rather than starting targets. This limitation is less severe early in a PCR reaction.
The data obtained during amplification is normalized by the processor which identifies a baseline of background signals (the expected signal in a PCR tube in the absence of a target nucleic acid) and which removes background signals from observed signals. The result of baseline subtraction is a measure of signal intensity which more accurately reflects the amount of target nucleic acid in a sample. Baseline subtraction calculations generally set the background signal observed in a tube during the cycles before amplification as the baseline. The range of cycles before amplification occurs is defined by endpoints (e.g., start and ending cycles) set by the user. Typical guidelines for selecting starting and ending cycles provide that the starting cycle is chosen after the typical variability in the first few cycles has abated and the ending cycle is chosen before amplification has occurred in any tube. Typically, signal obtained from these cycles is fitted with a line using a least mean squares algorithm. This best fit line is used to predict the background for all the cycles of a QPCR experiment, which is then subtracted from data generated from each sample which is being evaluated (see, e.g., as described in U.S. Pat. No. 5,928,907).
This system of setting a range for the calculation of a best-fit line has some limitations: 1) if samples with different starting quantities are used, then the best choice for the last endpoint will differ from sample to sample; 2) different users (or the same user at different times) can choose different endpoints; and 3) the same experiment analyzed with different endpoints will give a different result.
Inconsistency in baseline subtraction can cause larger errors in the final determination of unknown starting copies.